1. Field of the Invention
The present invention relates to transgenic plants having improved nutritional characteristics. More particularly, the present invention relates to transgenic plants, fruit and vegetable parts of which contain modified levels of sterol compounds, such as elevated levels of beneficial phytosterols, e.g., sitosterol, phytostanols, e.g., sitostanol, and esters thereof. Such transgenic plants can also contain elevated levels of tocopherols, such as α-tocopherol. In addition, these transgenic plants can contain reduced levels of campesterol and campestanol, and their respective esters, in their fruit and vegetable parts. Nucleic acid sequences encoding a variety of different enzymes that affect the biosynthesis and accumulation of sterol compounds and tocopherols in plants, and methods for using these sequences to produce such transgenic plants, are also provided. These methods comprise, for example, introducing a 3-hydroxysteroid oxidase such as a cholesterol oxidase, optionally in combination with a steroid 5α-reductase, and further optionally in combination with at least one tocopherol biosynthetic enzyme, into plants to elevate the levels of sitostanol and tocopherols, respectively, especially in seeds.
2. Description of Related Art
Phytosterols and Phytostanols
Phytosterols and phytostanols are well known to be beneficial for lowering serum cholesterol (Ling et al. (1995) Life Sciences 57: 195-206) and reducing the risk of cardiac disease. These compounds are poorly absorbed in the liver, and block the absorption of dietary cholesterol. Phytosterols and phytostanols, however, are present only in low amounts in seeds of dicotyledonous plants such as soybean, cotton, etc. Recently, strong evidence has been obtained demonstrating the role of phytostanols (hydrogenated forms of phytosterols, for example sitostanol) in reducing serum cholesterol in humans (Ling et al., supra). Ferulate and fatty acyl esters of sitostanol are naturally present in cereal grains in low levels (Seitz (1989) J. Agric. Food Chem. 37: 662-667; Dyas et al. (1993) Phytochem. 34: 17-29). In addition to phytosterols and phytostanols, grains and seeds also contain tocopherols and tocotrienols. Tocopherols act as antioxidants, and play a major role in protecting cells from damage caused by free radicals (Halliwell (1997) Nutrition Review 55: 44-60).
Insect-Resistant Transgenic Plants Expressing 3-Hydroxysteroid Oxidases
U.S. Pat. No. 5,518,908 discloses a method of controlling insect infestation in plants, comprising expressing a structural coding sequence encoding a 3-hydroxysteroid oxidase in cells of such plants, or in plant-colonizing microorganisms that can be applied to the plants, to impart insect resistance to the latter. In the case of transgenic plants, the goal was to provide monocotyledonous and dicotyledonous plants constitutively expressing an insecticidally effective amount of a 3-hydroxysteroid oxidase in plant parts such as leaves, flowers, and, in the case of cotton, bolls. The inventors expressed a preference for the use of constitutive promoters such as the nos, ocs, CaMV 19S and 35S, ssRUBISCO, and FMV 35S promoters to achieve this goal. Expression of the 3-hydroxysteroid oxidase in the cell cytoplasm, in extracellular spaces via the use of a secretory signal sequence, and in vacuoles and chloroplasts via the use of appropriate targeting sequences, is disclosed. However, no transgenic plants expressing a 3-hydroxysteroid oxidase transgene were produced. The invention disclosed in U.S. Pat. No. 5,518,908 is therefore distinctly different from that provided herein, as will become apparent from the description below.
U.S. Pat. No. 5,554,369, a divisional of the '908 patent, claims a method of controlling lepidopteran or boll weevil insect infestation of plants, comprising providing a 3-hydroxysteroid oxidase for ingestion by the insect.
U.S. Pat. No. 5,558,862, to the same inventors, claims a method of controlling insect infestation in plants by applying to the plant environment or plant seed a plant-colonizing microorganism that expresses heterologous DNA encoding a 3-hydroxysteroid oxidase.
U.S. Pat. No. 5,763,245, also to the same inventors, claims a method of controlling insect infestation in plants, comprising providing both a 3-hydroxysteroid oxidase and an insectidical Bacillus thuringiensis (Bt) protein for ingestion by lepidopteran insects. A method of producing a genetically transformed plant producing an insecticidally effective amount of a Bt protein and a 3-hydroxysteroid oxidase, comprising inserting into the genome of a plant cell a recombinant vector comprising nucleic acid sequences encoding the two proteins, as well as a promoter heterologous to the protein coding sequences which is effective to result in expression of the protein coding sequences in an insecticidally effective amount in a genetically transformed plant, is also claimed. As in their '908, '369, and '862 patents, supra, the inventors emphasize the use of constitutive promoters to provide uniform expression in the flowering portions of plants. Transgenic corn expressing either a Bt protein alone, or in combination with a 3-hydroxysteroid oxidase, i.e., cholesterol oxidase, is disclosed. Two populations of F1 generation plants expressing both proteins were produced by crossing plants subjected to a cholesterol oxidase transformation event with a plant subjected to a Bt transformation event.
Finally, European Pat. EP 0 706 320 B1 (corresponding to PCT International Publication WO 95/01098), also to the same inventors, and claiming priority from the same U.S. patent application from which the '908 patent issued, discloses transgenic tobacco expressing a 3-hydroxysteroid oxidase gene under the control of the constitutive FMV 35 promoter. As in the other patents discussed supra, the inventors again emphasized the use of plant constitutive promoters for expressing the 3-hydroxysteroid oxidase transgene to produce insect resistant plants.
Thus, a common feature of the disclosure of each of these patents is an emphasis on the use of a constitutive plant promoter to achieve expression of an insecticidally effective amount of a 3-hydroxysteroid oxidase in the flowering parts of plants to control insect infestation. Seed-specific, embryo-specific, and plastid-specific expression are neither disclosed nor suggested. Furthermore, no reason is given why such expression would be desirable, nor is any motivation provided therefor.
In addition to the foregoing patents, several reports relating to the expression of a 3-hydroxysteroid oxidase gene in transgenic plants have appeared in the technical literature. Corbin et al. (1994) Appl. Environ. Microbiol. 60: 4239-4244 discloses the cloning and expression of the insecticidal choM cholesterol oxidase gene from Streptomyces in E. coli, and transient expression thereof in tobacco protoplasts using the constitutive FMV 35S promoter. Cho et al. (1995) Appl. Microbiol. Biotechnol. 44: 133-138 discloses the expression of the Streptomyces cholesterol oxidase gene choA in transformed tobacco callus under the control of the constitutive CaMV 35S promoter. Corbin et al. (1996) HortScience 31: 699, Abstract No. 786, discloses the cloning and expression of a cholesterol oxidase gene in transgenic tobacco plants to yield plant tissue that exerted potent activity against boll weevils. Estruch et al. (1997) Nature Biotechnology 15: 137-141 is a review of approaches to pest control in transgenic plants, focusing primarily on Bacillus thuringiensis endotoxins. The use of cholesterol oxidases as insecticidal proteins is also discussed. The authors note that enzymatically active cholesterol oxidase was detected in extracts of tobacco protoplasts transformed with native cholesterol oxidase genes, citing the 1994 Corbin et al. and 1995 Cho et al. papers, supra. Discussing future directions in the area of insect resistant transgenic plants, the authors speculate on the use of “tighter tissue-specific promoters,” without giving any specific examples or suggestions. Jouanin et al. (1998) Plant Science 131: 1-11, another review article, focuses on the use of Bacillus thuringiensis δ-endotoxins and plant-derived genes such as those encoding enzyme inhibitors and lectins, to create insect resistant transgenic plants. The authors note the insecticidal activity of Streptomyces cholesterol oxidase genes, as well as the fact that most of the existing insect-resistant plants express a single resistance gene placed under the control of a constitutive promoter. In discussing strategies to retain insect susceptibility to B. thuringiensis genes expressed in transgenic plants, the authors note the use of constitutive, tissue-specific, and inducible promoters. They suggest that a means of avoiding the development of resistance by insects due to high selection pressure when constitutive toxin expression is employed in transgenic plants is via the use of tissue-specific promoters to limit insect exposure to the toxin in certain parts of the plant attacked by the insect. However, no specific examples or suggestions as to any particular tissues or tissue-specific promoters are disclosed. Interestingly, the authors note that targeted expression of insecticidal genes in transgenic plants could ensure public acceptance thereof, giving as an example the expression of an insect toxin in leaves of potato plants rather than in the tubers to control the Colorado potato beetle. This suggests toxin expression in plants only where it is needed to control insect pests, e.g., in non-food plant parts, when possible. Finally, Corbin et al. (1998) HortScience 33: 614-617 reviews strategies for identifying and developing new insecticidal proteins for insect control in transgenic crop plants. In addition to discussing Bacillus thuringiensis δ-endotoxins, the authors also review research on cholesterol oxidase. Without providing any experimental details, they note that they expressed the cholesterol oxidase gene from Streptomyces A19249 in transgenic tobacco, and demonstrated insecticidal activity of this tissue against boll weevil larvae. They also note that they are currently characterizing the expression and biological activity of cholesterol oxidase produced in transgenic cotton plants, again providing no experimental details.
Taken together, the foregoing patents and journal articles reveal that the approach generally employed up to this time to confer insect resistance on plants by recombinant methods has been to express an insecticidal protein constitutively in a transgenic plant. While suggesting that tissue-specific expression may have certain advantages, these publications provide no specific examples or strategies. Targeting of enzymes involved in insect resistance to plastids via the use of appropriate signal peptides in conjunction with constitutive promoters has been suggested. Note, for example, U.S. Pat. No. 5,518,908. The use of tissue-specific promoters, such as seed-specific promoters, for this purpose, and direct transformation of plastids, especially those in seed tissues, has not been disclosed or suggested. This literature does suggest, however, that limiting expression to plant parts attacked by insects, for example leaves, and avoiding expression in plant parts used as food or sources of food products or ingredients, for example potato tubers, is desirable. Thus, these references teach away from the concept of expressing an insecticidal protein such as a cholesterol oxidase in a plant part such as a seed, which can be a food, and a source of food products or ingredients such as oil and meal. Nor do any of these references teach or suggest the modification of endogenous phytostanol levels in plants transformed with such genes, or that such modification is even possible using such genes. Thus, these references provide no motivation to employ a cholesterol oxidase to alter phytosterol/phytostanol levels in plants, nor do they suggest that overexpression thereof in planta to modify phytosterol/phytostanol profiles carries with it a reasonable expectation of success.
Nutritional Value of Plant Oils
Vegetable and bran oils are the best natural sources of phytosterols and phytostanols. However, the amount of phytostanols in these oils is low relative to that of other sterol compounds. Increasing the content of phytostanols such as sitostanol in plant oils is thus desired in the art. Currently, most sitostanol is produced by processing soy oil, and converting β-sitosterol to sitostanol by hydrogenation. Such modifications are known to improve the anti-atherogenic activity of these phytosterols. However, besides adding cost, such chemical interventions can result in the formation of undesirable isomers. Therefore, modification of phytosterols by transesterification and/or reduction of double bonds in planta is an economical, efficient means of producing desired phytosterol derivatives, including phytosterol esters, phytostanols, and phytostanol esters. The ability to convert phytosterols to phytostanol esters naturally would add significant nutritional value to grains and seeds. Furthermore, naturally enhancing the levels of sitostanol, sitostanol esters, and tocopherols would not only improve the nutritional value of cereal grains and seeds, but also facilitates “stacking” of a combination of nutritionally important bioactive molecules in a single, convenient source. In this way, foods and food products containing bioactive molecules having superior bioavailability and efficacy can be designed to improve human nutrition and cardiovascular health.